Antisense oligonucleotides (AONs) are important tools for the specific modulation of gene expression. They are applied widely in the research laboratory for gene "knockdown" studies, and they are being developed clinically as therapeutics for a wide range of indications. The major challenge to more widespread implementation of this technology is cellular delivery. While many polymers and lipids have been used, with varying degrees of success, to deliver oligonucleotides to cells, their efficiency is highly variable depending on factors such as the chemistry and structure of the ON, the cell type used and the presence/absence of serum. Furthermore, the biophysical basis for delivery efficiency is poorly understood, and so development of delivery vectors remains a largely empirical process. Over the past several years, our group has pioneered a quantitative, engineering approach to oligonucleotide delivery. We have devoted considerable effort to identifying and quantifying the major barriers to cellular delivery and to developing relationships between the biophysics of polymer-oligonucleotide interactions and the extent and dynamics of antisense activity. Our work has highlighted the role of endosomal escape as a limiting cellular factor and competitive polyelectrolyte dissociation of polymer-oligonucleotide complexes as a major molecular biophysical factor governing antisense activity. We have found that the pH-sensitive polymer, poly(propylacrylic acid), PPAA, is able to mediate endosomal escape and improve delivery of AONs in conjunction with the cationic lipid, DOTAP. More recently, we have extended this concept to create novel graft copolymers that improve antisense-mediated gene silencing in the presence of serum. Our very promising Preliminary Results were based on a single composition of ethylene oxide/propylene oxide groups in the poly (oxyalkylene amine) grafted onto PPAA. In the proposed work, we will synthesize additional polymers that will provide insight into the effect of grafting density, poly (oxyalkylene amine) and acrylic acid chemistry on the physical and biological properties of the graft copolymers. Furthermore, we will continue to develop this novel antisense delivery system towards applications in cancer. Specifically, we will: (1) evaluate the effectiveness of our graft copolymers in several cell types with and without serum and (3) evaluate our delivery system in a tumor xenograft model.